It is increasingly evident that polymicrobial assemblages frequently underlie chronic infections, including those that occur in the lungs of cystic fibrosis (CF) patients and the wounds of diabetics. The complexity of such infections interferes with establishing etiology and complicates treatment. Clinical and in vitro data indicat that the Gram-negative Proteobacterium Pseudomonas aeruginosa is particularly adept at establishing itself and persisting within polymicrobial infections; however, a molecular explanation for this observation is lacking. The type VI secretion system (T6SS) is a complex intercellular effector protein delivery pathway. Though the system was initially thought to target host cells, during the prior award period our group discovered the primary function of the pathway is to deliver toxins in a cell contact-dependent manner between bacteria. Interestingly, P. aeruginosa possesses three non-redundant T6SSs, termed haemolysin co-regulated protein secretion islands I-III (H1-H3-T6SSs). Our studies revealed that the H1-T6SS of P. aeruginosa confers potent intra- and inter-species antibacterial activity mediated by its Tse (type VI secretion exported) effector substrates. In preliminary data provided in this proposal, we demonstrate that P. aeruginosa uses a far more elaborate interbacterial competition strategy than previously recognized. Indeed, we have discovered that P. aeruginosa employs at least two of its three T6SSs during interbacterial competition. Moreover, in addition to the Tse effectors, we have found that it possesses multiple members of a recently identified superfamily of T6S-delivered antibacterial phospholipases, termed the Tle (type VI secretion lipase effector) proteins. We propose herein to the test the hypothesis that the extensive and diverse T6S-dependent antibacterial repertoire of P. aeruginosa contributes to the fitness of the bacterium in the context of polymicrobial infections. In Aim 1 of this proposal, we will use lipidomic profilingto define the molecular targets of the recently identified Tle effectors and we will determine the capacity of these proteins to target clinically relevant bacterial species. In Aim 2, we evaluate the role of T6-dependent activity in vivo using a polymicrobial diabetic murine wound model. These experiments are structured such that the specific contribution of the antimicrobial activity of T6S effectors to disease outcome and in vivo fitness of P. aeruginosa during chronic infection can be ascertained. Finally, in Aim 3, we propose a secretion-independent, novel quantitative mass spectrometry-based approach for defining new T6S effectors. Effectors identified will be subject to phenotypic analysis and incorporated into our pipeline for evaluating the contribution of interbacterial T6S to competitive fitness in vivo. The studies outlined in this proposal stand t contribute significantly both to our basic understanding of the T6SS, and to the role that the pathway and its effectors play in polymicrobial infections.